The human immunodeficiency virus type 1 (HIV-1) is the causative agent of a national and international pandemic. No vaccine is currently available and viral variants resistant to current drug-based antiviral therapies are emerging world-wide. These facts make it imperative to identify new targets and strategies for development of anti-viral drugs. The gag gene of HIV and other retroviruses encode the viral structural precursor polyprotein, Gag, and is sufficient for assembly and release of virus-like particles. Many cellular proteins contribute to this process and represent potential novel targets. Our preliminary studies suggest that the inositol (1,4,5)-triphosphate receptor (IP3R) is involved in trafficking of HIV-1 Gag and the related avian Rous Sarcoma Virus (RSV) Gag to release sites on the plasma membrane. The IP3R protein has not been previously linked to retroviral trafficking. IP3R forms a ligand-gated calcium channel that regulates the release of intracellular calcium stores. Calcium release regulates many cellular processes, including intracellular trafficking and exocytosis. Inhibitors of IP3R interfere with Gag trafficking. Susceptibility to IP3R inhibitors requires the Late domains in the HIV and RSV Gag proteins, which are regions that bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 are cellular proteins that facilitate trafficking of HIV and RSV Gag, respectively, to sites on the plasma membrane where Gag assembles into virus-like particles (VLPs) and buds into the extracellular environment. The goals of the proposed studies are (AIM 1): To determine the role of IP3R in HIV Gag release;(AIM 2): To determine the role of IP3R in RSV Gag release;(AIM 3): To test for IP3R-Gag interaction in cells and define the relationship to proteins known to be involved in endocytic trafficking. Genetic, biochemical, and cell biological approaches will be used that will include targeted protein depletion using microinjection and short hybrid interfering RNAs;targeted depletion coupled to replacement with mutated IP3R proteins;use of IP3R antagonists and agonists;and fluorescence-based approaches. This investigation will potentially link endocytic trafficking of retroviral Gag proteins to calcium signaling and provide new opportunities for development of novel antiviral strategies that interfere with virus assembly and transmission.